There is currently a paucity of therapies for patients with advanced stages of thyroid cancer, and furthermore patients with anaplastic thyroid cancer continue to have one of the worst cancer prognoses with a 20% survival rate one year after diagnosis. The MAPK pathway accounts for a majority of the mutations that arise in thyroid cancer, with a high prevalence BRAF and Ras mutations. Therefore much effort has been devoted to targeting this pathway in the clinic; however, unlike melanoma, thyroid cancer appears to demonstrate primary resistance to MAPK pathway inhibition, and therefore limited efficacy has been observed at this stage. Our lab has discovered that Src is an effective target in thyroid cancer, observing decreased levels of proliferation, invasion, and metastasis in response to two structurally independent Src inhibitors, dasatinib (BMS-354,825) and saracatinib (AZD0530). We have recently focused our attention on dasatinib, because it is currently approved for the treatment of CML and PH+ ALL, and we are preparing the first clinical trial to test the efficacy of Src inhibition with dasatinib in advanced thyroid cancer patients. Evn though our preclinical data demonstrates strong therapeutic efficacy with dasatinib treatment, it is clear that resistance to single agent tyrosine kinase inhibitors is inevitable. In order to idenify mechanisms of resistance to Src inhibition, we have generated a panel of four thyroid cancer cell lines resistant to dasatinib (2 BRAF- and 2 RAS-mutant), with the goal of identifying key mechanisms of resistance that we can target in combination with Src to enhance the therapeutic efficacy. To begin to elucidate mechanisms of resistance, we performed genome wide RNA- sequencing on both the control and dasatinib-resistant cell lines, and identified that deregulation of the MAPK pathway and acquisition of the c-Src gatekeeper mutation (T341M) appear to be the key mechanisms mediating resistance to Src inhibition. Interestingly, MAPK pathway deregulation was present in both dasatinib- resistant BRAF- and RAS-mutant cell lines whereas acquisition of the c-Src gatekeeper was only present in the dasatinib-resistant RAS-mutant cell lines. Therefore the goals of my proposal are to determine the precise role of the MAPK pathway and the c-Src gatekeeper mutation in mediating resistance to dasatinib. Specifically in Aim 1, I will define the role of the MAPK pathway in Src inhibitor resistance; and in Aim 2, I will define how the MAPK pathway and the c-Src gatekeeper cooperate in mediating Src inhibitor resistance. This proposal emphasizes a molecular understanding of resistance mechanisms in response to Src inhibition that will provide the necessary framework for the development of new therapeutic strategies in order to combat and/or prevent resistance from occurring. The completion of the proposed research will help the National Cancer Institute fulfill their mission t support cancer research and training in the fundamental sciences.